The goal of this revised application is to determine the mechanisms by which sickle cell disease (SCD) increases inflammation and vasocongestion. Recently we reported that SCD increases circulating levels of High Mobility Group Box 1 (HMGB1) and that nearly 70% of the total TLR4 reporter activity in SCD plasma could be inhibited by anti-HMGB1 neutralizing antibodies. HMGB1 is a nuclear binding protein that aids in regulating gene expression and maintaining DNA structure. However, HMGB1 can also be released from activated neutrophils and injured cells and tissues. Once released, HMGB1 acts as a Damaged-Associated Molecular Pattern (DAMP) that, depending on its thiol oxidation status, activates the Receptor for Advanced Glycation Endproducts (RAGE) or Toll-Like Receptor 4 (TLR4). Although it is well-established that SCD increases neutrophil counts, neutrophil activation as well as tissue injury during vaso-occlusive crises (VOC), events that are all associated with HMGB1 release little is known about the role of HMGB1 in SCD. Here we hypothesize that SCD increases HMGB1 release which impairs EC function to increase vasocongestion. To test this hypothesis 3 aims are proposed. Aim 1 determines how SCD increases HMGB1 release and the impact this has on pathophysiology at baseline and after hypoxia\reoxygenation (H\R) injury. Sickle mice will be depleted of neutrophils and effects determined at baseline and after H/R injury with respect to HMGB1 activity and changes in levels of HMGB1, cf Hb and hemin. Aim 2 determines how HMGB1 impairs EC function with respect to vasodilatation, inflammation and adhesion. HMGB1 will be depleted in mice and effects of depletion determined with respect to vasodilatation; sVCAM-1, sICAM-1 and sE-selectin to assess EC injury and inflammation; and, with respect to WBC\RBC - EC interactions. Aim 3 determines the effects of oxidative stress in SCD on rates of HMGB1 thiol oxidation. The studies will determine how chronic states of oxidative stress in SCD mediate HMGB1 oxidation thiol status and the impact that HMGB1 oxidation status has on RAGE and TLR4 activity, inflammation and VOC. In vivo and ex vivo studies will reveal how targeting oxidative enzymes modulates HMGB1 oxidation status and isoform specific downstream signaling and pathophysiology. Studies will be performed in mice, in EC cultures and our RAGE & TLR4 reporter cell lines. The hypothesis tested is that oxidative stress in SCD accelerates oxidation of reduced HMGB1 to HMGB1 disulfide, establishing chronic states of TLR4 activation and increased IRF5 nuclear translocation, a potent signal for EC injury and death. These studies are designed to determine if and the extent to which the HMGB1-TLR4-IRF5 pathway is involved in the mechanisms by which SCD impairs EC function and increases vasocongestion. Our studies will provide some of the first evidence linking oxidative stress in SCD to the release and oxidation of HMGB1. Knowledge gained from our studies will help in the design of new therapeutic strategies for targeting HMGB1 to improve vascular health and limit vaso-occlusive crises in SCD.